Acrylamide gel electrophoresis of acidic mucopolysaccharides

ANALYTICAL

31, 51-58 (1969)

BIOCHEMISTRY

Acrylamide

Gel

Electrophoresis

Mucopolysaccharides J. C. HILBORN Department

‘I ’

AND P. A. ANASTASSIADIS

of Agticultural University,

of Acidic

Macdonald

Chemistry, College

Maedonald College of McGill P.O., Quebec, Canada

Received February 4, 1969

It has been suggested that polyacrylamide gel is not a satisfactory supporting medium for the electrophoretic separation of acidic mucopolysaccharides because the sieving effect of the medium is a disadvantage for substances of polydisperse nature such as the mucopolysaccharides, as it does not permit their separation solely on the basis of charge density (1). Kimura et al. (2) have reported, however, that chondroitin sulfates A and B and heparin were separated by polyacrylamide gel electrophoresis with 0.05 M formate buffer, although their published figures showed an overlapping of bands which did not indicate a satisfactory separation of a mixture of mucopolysaccharides. Electrophoresis of acidic mucopolysaccharides carried out in our laboratory at several pH values over the whole effective buffer range of formic acid/sodium formate solution (pH range 2.6-3.4) gave unsatisfactory results. Satisfactory separation was obtained at pH 11.5 when gels were prepared in a 0.1 M phosphate buffer, and compactness of bands was improved with the addition of sodium formate to the buffer. The effects of other buffers and conditions of electrophoresis on the electrophoretic patterns of mucopolysaccharides were investigated. APPARATUS,

GEL PREPARATION,

AND MATERIALS

The apparatus used was based on the original design by Raymond and Wang (3) which had been tried extensively in this laboratory (4). The water-cooled gels (18 x 10 x 0.5 cm) were run horizontally. Gels were prepared by dissolving the desired amount of Cyanogum-41 (American Cyanamide Company) and 1 This work was supported by National ResearchCouncil of Canada Grant A-2005. 2 Macdonald CollegeJournal Series No. 597. 51

52

HILBORN

AND ANASTASSIADIS

0.2 gm of ammonium persulfate in 100 ml of buffer. This solution was filtered, and after 1.0 ml of dimethylaminopropionitrile was added, it was poured into the gel form, covered with Plexiglas and held firm with lead weights until the gel was ready. Samples dissolved in buffer were soaked up in 1 x 0.3 cm strips of Whatman No. 3 filter paper and inserted in the gel. A strip was saturated with approximately 0.01 ml of solution. The electropherograms were stained with a 0.5% solution of Alcian Blue in 3 “/o acetic acid and were destained with 3% acetic acid. The following reference acidic mucopolysaccharides were used : Heparin-calcium salt (grade III) ; chondroitin sulfate-sodium salts of mixed isomers (grade III) (Sigma Chemical Company, St. Louis, MO.) ; hyaluronic acid (Nutritional Biochemicals Corporation, Cleveland, Ohio) ; and chondroitin sulfates A, B, C, and D-sodium salts (Miles Laboratories Incorporated, Elkhart, Ind.) . Biological materials, prepared as described below, were used in order to test the efficiency of the resolution of the procedure when preparations of the polymers are obtained from tissues. Crude acidic mucopolysaccharides were isolated from avian (rooster) skin, testes, comb, and wattles following papain digestion and precipitated with cetylpyridinium chloride as described by Scott (5). Mucopolysaccharides were isolated from sheep lung after hydrolysis with pepsin and trypsin, precipitated with 1.2 volumes of alcohol, purified with amyl alcohol/chloroform and Lloyd reagent and fractionally reprecipitated with alcohol according to the method of Meyer et al. (6). RESULTS

AND

DISCUSSION

In the publication of Kimura et al. (2)) the pH and voltage used were not mentioned and therefore a number of electrophoreses were performed by us with formate buffer of different pH values between 2.6 and 3.4. Short migrations and poor separations were obtained at all the pH values tried in the above range. Figure 1 illustrates the pattern obtained after electrophoresis with formate buffer at pH 3.0 (under conditions described in Table la). Heparin exhibited the fastest mobility whereas hyaluronic acid remained at the origin. In spite of the poor results, the compactness of the bands was taken as an indication that the method had some potentialities. Electrophoreses with modified conditions as regard voltages, ionic strengths of the formate, pH values, and an increase in gel concentration, to decrease pore size and sharpen the bands, were therefore attempted.

ELECTROPHORESIS

OF MUCOPOLYSACCHARIDES

53

FIG. 1. Acrylamide gel electrophoresis of mucopolysaccharides with formate buffer, pH 3.0 (Table la) : (1 and 5) mixture of heparin, chondroitin, sulfate (mixed isomers), and hyaluronic acid; (2) hyaluronic acid; (3) chondroitin sulfate (mixed isomers) ; (4) h ep arin. All samples applied at cathode (top of photograph).

Electrophoreses performed under conditions indicated in b of Table 1 gave satisfactory separation but poor compactness, which was improved when the electrophoreses were performed under conditions indicated in c and d. Figure 2 gives the pattern obtained under conditions indicated in d. It is obvious that under these conditions mucopolysaccharides could be made to migrate 1

2

3

FIG. 2. Acrylamide gel electrophoresis of Cyanogum was dissolved in a solution of (Table Id). 1 to 5 as in Figure 1. A, zone B, zone of migration of chondroitin sulfate;

4

5

mucopolysaccharides at pH 11.5. 0.1 M formic acid and NaOH of migration of hyaluronic acid; C, zone of migration of heparin.

k

M M M M

M

M 0.1 M 0.1 M 0.1 M 0.1 M 0.1 M 0.075 M 0.1 M 0.1 M 0.125 M 0.250 M

0.01

0.01 M 0.1 M 0.01 M 0.1 M

0.05 0.1 0.1 0.1 0.1

Kind

6% Cyanogum

1

100 100

11.4 11.5

110

80

80

34

34

8 122 72 40 50 50

Il-lA

Good

7

was used.

Fair

Fair

Good

Fair

Poor Good Good Good Good Fair

Mobility

7

7

7

6

Time, hr

Good

Fair

Good

Good

Good

Poor Good Good Good Good Good

Separation

Results

of Acidic Mucopolysaccharides

was used except in a where 50/, Cyanogum

100

100

11.5 11.4

100

150 200 150 100 150 100

Volts

Conditions

Investigated for Separation in Polyacrylamide Gel*

11.5

3.0 11.5 11.5 11.5 10.5 11.5

PH

of Electrophoresis

HCOOH/NaOH HCOOH/NaOH HCOOH/NaOH HCOOH/NaOH HCOOH/NaOH HCOOH Na3P04 HCOOH Na,POI HCOOH Na3P04 Na,PO, Na*HPOr HCOONa Na3P01 NatHPOa HCOONa Na3P04 NazHPOr HCOONa (bridge) HCOONa (gel)

* In all the electrophoreses

j

i

h

9

d ;

C

b

a

No.

BUtk

Buffers and Conditions

TABLE

Good

Fair

Good

Good

Good

Good Poor Fair Good Poor Good

Compactness

z b 2 % g E g ;3

x F: 8 !z

ELECTROPHORESIS

OF MUCOPOLYSACCHARIDES

55

a sufficiently long distance while maintaining a level of compactness suitable for separation of the mixture. Figure 3 gives the patterns obtained when electrophoresis of mucopolysaccharides isolated from sheep lung at the 40, 50, and 60% ethanol levels (6) was carried out under conditions d. Lowering the pH to 10.5 under the conditions indicated in e resulted in bands of inferior compactness. As it became evident that the pH value of 11.5 was a critical factor in the successful separation of the mucopolysaccharides, a buffer was tried that would function at pH 11.5. The addition of sodium phosphate to the formate under conditions indicated in f, g, and h gave results comparable to those achieved with conditions d. Only the duration of the run had to be altered. Borate buffers at pH 3.6 and 9.6 did not give satisfactory separations. Phosphate buffers of various concentrations between 0.01 and 0.1 M were used at pH values between 7.0 and 11.7 with and without formate. The most significant results of these experiments are the following. Electrophoreses with a phosphate buffer, pH 11.4, to which sodium formate had been added (conditions i) gave bands with markedly improved sharpness, which permitted the separation of a mixture of mucopolysaccharides (Figure 4). A decrease of the formate concentration, as indicated in j, had an adverse effect on the sharpness of the bands while an increase 1

2

3

4

FIG. 3. Acrylamide gel electrophoresis of mucopolysaccharides at pH 11.5. Cyanogum was dissolved in a solution of 0.1 M formic acid and NaOH (Table Id). (1) Mixture of hyaluronic acid, chondroitin sulfate (mixed isomers), and heparin; (2) mucopolysaccharides isolated from sheep lung at the 40% ethanol level (6) ; (3) mucopolysaccharides isolated from sheep lung at the 50% ethanol level (6) ; (4) mucopolysaccharides isolated from sheep lung at the 60% ethanol level (6).

56

FIG. 4. phosphate conditions isomers),

HILBORN

AND

ANASTASSIADIS

Acrylamide gel electrophoresis of mucopolysaccharides with 0.1 M buffer (pH 11.4) in the presence of 0.1 M sodium formate under described in Table li: (1) mixture of chondroitin sulfate (mixed and heparin; (2) chondroitin sulfate (mixed isomers) ; (3) heparin.

of the formate concentration, as indicated in k, enhanced the compactness of the bands. When the buffer indicated in k was used to prepare a gel but the Cyanogum content was reduced from 6 to 5%, the gel was found to be fragile and difficult to handle. Streaking of bands was evident when the Cyanogum concentration was raised to ‘7%. In all cases mentioned so far, electrophoresis was carried out using continuous buffer systems. However when the buffer system indicated in k was used in the troughs, but buffer of double concentration of formate was used in the gel, better separation of a mixture of mucopolysaccharides was achieved (Fig. 5). Under these conditions a mixture containing hyaluronic acid, chondroitin sulfates B and C, and heparin could be separated. Chondroitin sulfates A and B had the same mobility. Hyaluronic acid was not observed to migrate. The chondroitin sulfate D band was observed to overlap the bands produced by the B and C or A and C isomers (however this was not considered a serious defect in the method as the D species had been found only in shark cartilage (7) to date). The method is sensitive to the extent of lo-20 pg of each mucopolysaccharide. Figure 6 shows the patterns obtained when mucopolysaccharides from biological materials were subjected to electrophoresis. Mucopolysaccharides isolated from avian skin were resolved to three distinct fractions, one with mobility corresponding to hyaluronic acid, a second with mobility corresponding to chondroitin sulfate

ELECTROPHORESIS 123

OF MUCQPOLYSACCHARIDES 456

57

7

FIG. 5. Acrylamide gel electrophoresis of mucopolysaccharides with phosphate buffer (pH 11.5) in the presence of sodium formate under conditions described in Table lk: (1) mixture of hyaluronic acid, chondroitin sulfate B, chondroitin sulfate C, and heparin; (2) hyaluronic acid; (3) chondroitin sulfate D; (4) chondroitin sulfate C; (5) chondroitin sulfate B; (6) chondroitin sulfate A; (7) heparin.

B, and a third with mobility greater than any of the reference mucopolysaccharides used. Mucopolysaccharides isolated from testes appeared to contain a single component with mobility corresponding to the fast moving component of avian skin. Mucopolysaccharides isolated from avian comb were resolved to two fractions, a major one with a mobility corresponding to hyaluronic 123

Le

45

__I

6

7

1 d

FIG. 6. Acrylamide gel electrophoresis of mucopolysacharides under conditions described in Table lk: (1) mixture of hyaluronic acid, chondroitin sulfate B, and heparin; (2) chondroitin sulfate C; (3) mucopolysaccharides isolated from avian skin (5) ; (4) mucopolysaccharides isolated from avian testes (5) ; (5) mucopolysaccharides isolated from avian comb (5) ; (6) mucopolysaccharides isolated from avian wattles (5) ; (7) heparin.

58

HILBORN

AND ANASTASSIADIS

acid and a minor one with mobility corresponding to chondroitin sulfate C. Finally, mucopolysaccharides isolated from wattles were resolved to two components, one with mobility corresponding to hyaluronic acid and a second, in a small amount, with a mobility similar to the mobility of the fast moving component obtained from skin and testes. REFERENCES 1. HORNER, 2. KIMURA,

A. A., Can. J. Biochem. 45, 1009 (1967). A., WATANABE, T., AND NAGAI, Y., Fuhxshima ‘71 (1964). 3. RAYMOND, S., AND WANG, Y., Anal. Biochem. 1, 391 (1960). 4. GRANT,

D.

L.,

MARTIN,

W.

G.,

242, 3912 (1967). 5. SCOTT, J. E., Methods Biochem. 6. MEYER,

K.,

LINKER,

A.,

AND

ANASTASSIADIS,

P.

J. Med. Sci. 11,

A.,

J. Biol.

Chem.

Anal. 8, 145 (1963).

DAVIDSON,

E. A., AND

WEISSMAN,

B.,

205, 611 (1953). 7. BRIMACOMBE, J. S., AND WEBBER, J. M., “Mucopolysaccharides” Library, Vol. 6), American Elsevier, New York, 1964.

J. Biol. Chem. (B.B.A.